This program project focuses on the structure and function of tumor blood vessels from many angles: roles and functions of angiogenic cytokines and endogenous inhibitors in tumors and in surrogate tumor blood vessels, the gene expression induced by these factors and their role in tumor angiogenesis, and the impact of angiogenic cytokines and angiogenesis on tumor progression. This proposal outlines a series of experiments that focus on one of major signaling pathways downstream of the major angiogenic cytokines, the Akt pathway, as a mediator of many tumor blood vessel abnormalities: their large size, poor support, increased permeability and instability. Our preliminary data demonstrate that Akt signaling in physiological angiogenesis involves a very precise switching 'on' and 'off of the Akt signal. However, when this 'off switch is inhibited, the resulting vessels recapitulate many aspects of tumor blood vessels, vessels of other vascular anomalies and 'mother vessels', a term coined by Dr. Dvorak to describe blood vessels formed by overexpression of VEGF-A and other cytokines. This grant application seeks to explore the role of Akt in 'mother vessel'/'tumor vessel' formation, and the impact of downstream Akt targets on the individual features and functions of these abnormal blood vessels. We also propose to explore the mechanism behind our novel finding that TSP-1 modulates Akt signaling. This project is written for 3 years to enable it to fold into the existing funding schedule of this program project. Aims: 1. Test the hypothesis that upregulation of Akt activity is sufficient to recapitulate the structure and function of abnormal blood vessels reminiscent of tumor vessel in a non-tumor environment 2. Identify the downstream signaling pathways that mediate Akt affects 3. Explore the mechanisms of Thrombospondins on Akt signaling. [unreadable] [unreadable] [unreadable] COLLABORATING INSTITUTIONS (S): Massachusetts General Hospital, Boston, MA [unreadable] Children's Hospital, Boston, MA [unreadable] [unreadable] [unreadable] PROGRESS DURING THE CURRENT FUNDING PERIOD: This program has now completed its second year. During this period 17 publications have resulted from program related studies. A number of these have appeared in high impact journals and 11 list multiple authors who are part of this Program Project. Overall progress has been excellent. Project 1, "Structure and Function of New Blood Vessels," is directed by Dr. Harold Dvorak, in conjunction with Dr. Laura Benjamin. The studies under Specific Aim 1 where the investigators characterized new blood vessel induction by VEGF-A versus PIGF, VEGF-A/PIGF heterodynamers and FGF, are now completed and the work published in Nature Medicine. Specific Aim 2 is to employ inducible gene expression in mammary tumors to overexpress or not express the angiogenic factors studied under Specific Aim 1. These studies are underway and have resulted in two publications. Specific Aim 2 will use chip technology to profile genes that are differentially expressed in 'mother' vessels in response to local overexpression of VEGF-A or PIGF. Several novel genes have been characterized in the first two years of the project and many other potentially important genes also identified. Two manuscripts are in preparation describing this work. [unreadable] [unreadable] Project 2, Signaling Pathways that Determine Vascular Structure and Function, is the newly proposed project in this supplement and is considered in detail below. It is noted that Dr. Benjamin is a co-author of six of the 17 publications listed, several of which are very relevant to proposed supplement Project 2. [unreadable] [unreadable] Project 3, "Role of Placental Growth Factor in Normal and Neoplastic Skin Angiogenesis," is led by Dr. Michael Detmar. Specific Aim 1 is a study of the role of PIGF in skin vasculaturation and in experimental skin inflammation employing transgenic animal models. A novel transgenic mouse model for the targeted overexpression of human PIGF-2 in basal epidermal keratinocytes and other cells has been made. Studies of these animals have resulted in a paper published in Blood. Specific Aim 2 will employ these models to study chemically-induced skin cancer. Initial results suggest that PIGF plays a role in epithelial tumorigenesis. Specific Aim 3 will employ transfected PIGF and semaphorin D gene constructs to study the effects of modulating angiogenesis on the growth of human squamous cell carcinoma xenotransplants. Stably transfected cell lines have been obtained to pursue this aim and data are now being collected on these models. [unreadable] [unreadable] Project 4, "Inhibition of Angiogenesis by Thrombospondin-1," is led by Dr. Jack Lawler with the participation of Dr. R. Khosravi-far and Dr. S. Parangi. Specific Aim 1 seeks to characterize the CD36 receptor complex on endothelial cells to which TSP-1 binds. Specific Aim 2 will study mechanisms involved in TSP-1-induced apoptosis. Work is progressing on these aims, but appears quite preliminary. Dr. Lawler is co-author of three papers that report work on the ligand TSP-1 which have appeared in high impact journals. [unreadable] [unreadable] In addition, there are four cores (Administration, Morphology, Cell Biology, and Genomics) which are all operational and fulfill their role in the overall Program Project. Overall progress, as noted above, has been excellent. [unreadable] [unreadable] [unreadable] [unreadable]